The Mechanism of Oxidative Stress Stabilization of the Thromboxane Receptor in COS-7 Cells

Abstract

The 8-iso-prostaglandin F(2alpha), a prostanoid produced in vivo by cyclooxygenase-independent free-radical-catalyzed lipid peroxidation, acts as a partial agonist on the thromboxane receptor (TXA(2)R) and is a potent vasoconstrictor in the oxidatively stressed isolated perfused rat heart. We hypothesized that the response in the isolated heart may be due to augmentation of TXA(2)R density, which may be initiated by the presence of oxidative radicals. Previous studies have shown that TXA(2)R density is increased during atherosclerosis on both the medial and intimal smooth muscle layers in human coronary arteries. Here we describe the effect of oxidative stress on TXA(2)R. The thromboxane A(2) receptor beta isoform (TXA(2)Rbeta) was transiently expressed in COS-7 cells. Immunofluorescence suggested that the presence of H(2)O(2) increased translocation of TXA(2)Rbeta from the endoplasmic reticulum (ER) to the Golgi complex. H(2)O(2) treatment also increased binding of a TXA(2)R antagonist ([(3)H]SQ29548) to membranes. Degradation kinetics of TXA(2)Rbeta following cycloheximide treatment, a protein synthesis inhibitor, suggested not only that TXA(2)Rbeta is a short-lived protein predominantly localized to the ER but also that TXA(2)Rbeta degradation is modulated in the presence of H(2)O(2). Our results indicate that oxidative stress induces maturation and stabilization of the TXA(2)Rbeta protein probably by intracellular translocation. Importantly, these observations also suggest that TXA(2)Rbeta levels are modulated by ER-associated degradation and controlled by the efficiency of transport to post-ER compartments. Stabilization of the TXA(2)Rbeta by translocation from a degradative compartment, i.e. the ER, can account for the augmentation of receptor density observed in vivo.