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

Abstract

The 8-iso-prostaglandin F 2␣ , 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 ␤ isoform (TXA 2 R␤) was transiently expressed in COS-7 cells. (TXA 2 ) 1 is an unstable arachidonate metabolite, implicated as a mediator in diseases such as myocardial infarction, stroke, and bronchial asthma (1). Binding of TXA 2 to its receptor, a polytopic membrane-spanning protein, induces vasoconstriction and platelet aggregation, as well as mitogenesis and hypertrophy of vascular smooth muscle cells (2). Two TXA 2 receptor (TXA 2 R) isoforms have been identified, TXA 2 R␣ (343 amino acids), which is mainly located in the placenta, and TXA 2 R␤ (407 amino acids), located in the endothelium; these isoforms are generated by the alternative splicing of a single gene (3, 4). The TXA 2 R is part of the G proteincoupled receptor superfamily, and evidence suggests that TXA 2 -induced production of second messenger inositol polyphosphates results from the activation of the G q11 family of heterotrimeric G proteins (5).Isoprostanes are formed by free radical attack on membrane phospholipids during oxidative stress (6). They are found in increased concentration in patients with coronary heart disease and are potent vasoconstrictors (7). We have shown that one of these, the 8-iso-prostaglandin F 2␣ , is a potent coronary vasoconstrictor, and its effect is exerted via partial agonist action on the TXA 2 R (8). This mechanism of action on TXA 2 R, in vascular smooth muscle and in platelets, has been confirmed in a TXA 2 R knock-out mouse (9). Our data suggested that a critical determinant of the intrinsic activity of the isoprostane is the TXA 2 R reserve, and this has subsequently been supported by another study (10). We have shown that, in the normal rat heart perfused at constant pressure in the Langendoff mode, 8-iso-prostaglandin F 2␣ had no effect, even though U46619, a TXA 2 R agonist, produced a pronounced vasoconstriction. However, after an oxidative stress induced by 30 min of low flow and reperfusion or by a superoxide-generating system (i.e. xanthine and xanthine oxidase), 8-iso-prostaglandin F 2␣ became a potent vasoconstrictor, whereas the response to U46619 was unchanged (11). Responses to both agonists were inhibited by the TXA 2 R antagonist SQ29548, suggesting that they act upon the same receptor.The rapidity of the change in response suggests that this is unlikely to be due to alterations in gene expressio...

Interaction of angio-associated migratory cell protein with the TPα and TPβ isoforms of the human thromboxane A2 receptor

“…However, a discrepancy exists in the literature between the deduced primary sequence of AAMP [1] and that sequence (NM_001087) deposited at the National Center for Biotechnology Information As an additional means of examining the novel interaction, confocal image analysis was also used to investigate the expression and possible co-localization of TPα and TPβ with AAMP endogenously expressed in the HEK.TPα and HEK.TPβ clonal cell lines. Consistent with previous reports [61], while TPα is almost exclusively expressed at the plasma membrane, TPβ differs in that it is expressed both at the plasma membrane but also as an intracellular reserve associated with the endoplasmic reticulum ( Figure 2E, anti-HA panels). Immunostaining of endogenous AAMP in the latter cell lines was examined using an affinity purified antibody directed to the C-terminal residues of AAMP (~ 334 -434; Strategic Diagnostics Incorporated) and established that it is expressed both at the plasma membrane and in the cytosolic fraction where it displayed extensive punctate staining, possibly associated with a fibrous network ( Figure 2E, anti-AAMP panels).…”

Upregulation of Heme Oxygenase 1 by Hemin Impairs Endothelium-Dependent Contractions in the Aorta of the Spontaneously Hypertensive Rat

“…This may be associated with the antioxidant effect of HO-1, because ROS enhance the stability and increase the density of functional TP receptors at the cell membrane. 7,42,43 …”

The G12 Family of G Proteins as a Reporter of Thromboxane A2 Receptor Activity

“…We suspect that, in vivo, the actions of 8-iso-PGF 2␣ through TP␣ would require significant receptor reserve or the aforementioned accumulation of activated G 13 as a result of activation outpacing deactivation. It is interesting to note that dynamic changes in TP levels, perhaps influencing responsiveness to 8-iso-PGF 2␣ , have been argued to occur in pathophysiological states, including oxidative stress (Valentin et al, 2004). Signaling for 8-iso-PGF 2␣ through the G 12 family of G proteins may not be evident in cells where G 12 predominates as a transducer.…”