9246605

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3

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 Kd of 13.84 +/- 2.58 nM and Bmax of 3.54 +/- 0.28 pmol/10(6) 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 (Bmax) decreased 31.61% and 34.8% by the pretreatment with cholera toxin (200 micrograms/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 intracellular 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.

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0929-7855

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155-69

pubmed-meshheading:9246605-8,11,14-Eicosatrienoic Acid, pubmed-meshheading:9246605-Binding Sites, pubmed-meshheading:9246605-Bucladesine, pubmed-meshheading:9246605-Cell Adhesion, pubmed-meshheading:9246605-Cell Line, pubmed-meshheading:9246605-Cholera Toxin, pubmed-meshheading:9246605-Cyclic AMP, pubmed-meshheading:9246605-Cyclic AMP-Dependent Protein Kinases, pubmed-meshheading:9246605-Down-Regulation, pubmed-meshheading:9246605-Enzyme Activation, pubmed-meshheading:9246605-Enzyme Inhibitors, pubmed-meshheading:9246605-Humans, pubmed-meshheading:9246605-Isoquinolines, pubmed-meshheading:9246605-Monocytes, pubmed-meshheading:9246605-Pertussis Toxin, pubmed-meshheading:9246605-Protein Binding, pubmed-meshheading:9246605-Receptors, Cell Surface, pubmed-meshheading:9246605-Signal Transduction, pubmed-meshheading:9246605-Stereoisomerism, pubmed-meshheading:9246605-Sulfonamides, pubmed-meshheading:9246605-Virulence Factors, Bordetella

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

Journal Article, Research Support, U.S. Gov't, P.H.S.