Post-receptor signal transduction and regulation of 14(R),15(S)-epoxyeicosatrienoic acid (14,15-EET) binding in U-937 cells.
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.
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
Shen
S Y
SY
Belosludtsev
Y Y
YY
Falck
J R
JR
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D003994:Bucladesine / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D009000:Monocytes / Q000378:metabolism*; D037342:Pertussis Toxin; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism; D015398:Signal Transduction; D013237:Stereoisomerism; D013449:Sulfonamides; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007546:Isoquinolines; D011956:Receptors, Cell Surface; D013449:Sulfonamides; D010566:Virulence Factors, Bordetella; D003994:Bucladesine; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D002772:Cholera Toxin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D017868:Cyclic AMP-Dependent Protein Kinases; D015126:8,11,14-Eicosatrienoic Acid; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide