Characterization of the vascular thromboxane A2/prostaglandin endoperoxide receptor in rabbit aorta. Regulation by dexamethasone

Abstract

Recently, we have shown that dexamethasone treatment of rabbits specifically reduces vascular smooth muscle responsiveness to agonists that interact with the vascular thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. One potential site at which dexamethasone can influence prostanoid-mediated vasoconstriction may be at the level of the vascular TXA2/PGH2 receptor. Therefore, we characterized the vascular TXA2/PGH2 receptor in rabbit aortic membranes and examined the influence of dexamethasone treatment on vascular TXA2/PGH2 receptor affinity and number. The binding of [125I][1S-(1α,2β(5Z),3α(1E,3R)4α)]-7-[3-(3-hydroxy-4- (p-iodophenoxy)-1-butenyl)-7-oxabicyclo[2.2.1]heptan-2-yl]-5- heptanoic acid ([125I] BOP), a potent TXA2/PGH2 receptor agonist, to rabbit aortic membranes was saturable displaceable, and dependent on protein concentration. Scatchard analysis of equilibrium binding data disclosed one class of high affinity binding sites with a K(d) of 0.44 ± 0.13 nM and a B(max) of 114.4 ± 5.2 fmol/mg protein (n = 7). Removal of the endothelium before membrane preparation did not significantly alter the affinity or number of binding sites for [125I]BOP. Kinetic analysis of the rates of [125I]BOP association/dissociation yielded a K(d) of 0.62 nM. The ability of various agonists at the TXA2/PGH2 receptor to displace [125I]BOP from vascular membranes correlated well with their contractile potencies in rabbit aortic rings. Moreover, stereospecific displacement of [125I]BOP binding in aortic membranes and inhibition of U46619-mediated aortic contractions were obtained with the stereoiomers L657925(-) and L657926(+). Collectively, these data suggest that this binding site represents the functionally relevant vascular TXA2/PGH2 receptor. In functional experiments, [127I]BOP induced concentration-dependent contractions of the rabbit aorta, which were reduced by 52% in vessels from dexamethasone-treated rabbits. Binding experiments performed in aortic membranes from dexamethasone-treated rabbits revealed a 25% reduction in vascular TXA2/PGH2 receptor number with no change in affinity. Thus, the dexamethasone-induced decrease in TXA2/PGH2 receptor number in aortic membranes from dexamethasone-treated rabbits may contribute to the accompanying decrease in vascular responsiveness to TXA2/PGH2 receptor agonists.