Elsevier

Biochemical Pharmacology

Volume 75, Issue 12, 15 June 2008, Pages 2301-2315
Biochemical Pharmacology

Characterization of isoprostane signaling: Evidence for a unique coordination profile of 8-iso-PGF with the thromboxane A2 receptor, and activation of a separate cAMP-dependent inhibitory pathway in human platelets

https://doi.org/10.1016/j.bcp.2008.03.014Get rights and content

Abstract

Since isoprostanes are thought to participate in the pathogenesis of thrombosis, presumably through their interaction with thromboxane receptors (TPRs), we examined the ability of 8-iso-PGF to bind/signal through TPRs. Using TPR expressing HEK cells, it was found that 8-iso-PGF mobilized calcium and bound TPRs with a dissociation constant (Kd) of 57 nM. Interestingly, site-directed-mutagenesis revealed that 8-iso-PGF has a unique coordination profile with TPRs. Thus, while Phe184 and Asp193 are shared by both 8-iso-PGF and classical TPR ligands, Phe196 was found to be required only for 8-iso-PGF binding. Functional studies also revealed interesting results. Namely, that 8-iso-PGF signals in human platelets through both a stimulatory (TPR-dependent) and an inhibitory (cAMP-dependent) pathway. Consistent with the existence of two signaling pathways, platelets were also found to possess two separate binding sites for 8-iso-PGF. While the stimulatory site is represented by TPRs, the second cAMP inhibitory site is presently unidentified, but does not involve receptors for PGI2, PGD2 or PGE2. In summary, these studies provide the first documentation that: (1) 8-iso-PGF coordinates with Phe184, Asp193 and Phe196 on platelet TPRs; (2) Phe196 serves as a unique TPR binding site for 8-iso-PGF; (3) 8-iso-PGF signals through both stimulatory and inhibitory pathways in platelets; (4) 8-iso-PGF inhibits human platelet activation through a cAMP-dependent mechanism; (5) 8-iso-PGF interacts with platelets at two separate binding sites. Collectively, these results provide evidence for a novel isoprostane function in platelets which is mediated through a cAMP-coupled receptor.

Introduction

The family of eicosanoids called isoprostanes possess a chemical structure that is isomeric to the classical prostanoids [1]. While the prostaglandins are produced as a result of cyclooxygenase enzyme activity, isoprostanes are generally thought to form nonenzymatically by free radical-mediated peroxidation of arachidonic acid (AA) [2]. Separate evidence has suggested that cyclooxygenase activity may also contribute to isoprostane production in selected tissues [3], [4]. Of the isoprostane members, 8-iso-PGF appears to be the most abundantly produced in vivo[1], and has been the most extensively studied.

After their initial identification, evidence was provided that isoprostanes may be involved in the development of certain disease states. For example, results obtained with an in vivo mouse model suggested that isoprostanes participate in thrombus development at sites of vascular injury [5]. Other studies found that the levels of isoprostanes become elevated in patients with atherosclerosis [6], acute myocardial infarction [7], as well as in a canine model of ischemia-reperfusion injury [7]. Regarding a possible role for isoprostanes in human brain disorders, it has been shown that isoprostanes are associated with the pathogenesis of Alzheimer's disease [8]. Finally, in vitro studies revealed that isoprostanes can induce oligodendrocyte progenitor cell death [9].

Due to the potential role of isoprostanes in the pathogenesis of disease, their cellular signaling pathways and biological effects have been under investigation. In this connection, isoprostanes were found to exert their biological activity in many cell types, e.g., platelets, vascular smooth muscle, kidney, etc., through TPR activation. For instance, it was found that 8-iso-PGF caused platelet activation (shape change and reversible aggregation), that was sensitive to TPR antagonism [10], [11]. In addition, a TPR antagonist was shown to block 8-iso-PGF-induced vasoconstriction of vascular smooth muscle cells [12], carotid arteries [10], and renal glomeruli [13]. While recently disputed [10], the existence of discrete stimulatory isoprostane receptors in smooth muscle cells and human platelets has also been proposed [14], [15], [16], [17]. This suggestion was based on differences between the potencies of 8-iso-PGF and TPR agonists in inducing DNA synthesis [14] and MAP-kinase activation [15]. In summary, there are clear inconsistencies concerning the mechanisms by which isoprostanes modulate cellular function. Based on these considerations, we investigated three aspects of isoprostane biology: (1) their molecular interaction and signaling through TPRs; (2) their potential signaling through TPR-independent pathways; (3) their functional effects on human platelets and the mechanisms by which they produce these effects.

The initial experiments performed a comprehensive characterization of the interaction between 8-iso-PGF and human TPRs using mutagenesis studies. Using 19 different TPR mutant cell lines, our data identified three key residues, i.e., Phe184, Asp193, and Phe196 that are critical for 8-iso-PGF binding. These data also revealed a coordination site between 8-iso-PGF and TPRs, i.e., Phe196, that is unique for 8-iso-PGF. Moreover, our results provide evidence that human platelets possess two 8-iso-PGF binding sites, which is consistent with the finding that 8-iso-PGF signals through two separate pathways (one stimulatory and one inhibitory). While the stimulatory signaling pathway is TPR-dependent, the inhibitory pathway is TPR-independent and associated with elevation of platelet cAMP levels.

Section snippets

Reagents

The Fura2/AM dye was from Molecular Probes (Eugene, OR). [3H]8-iso-PGF, 8-iso-PGF, and other prostaglandins were from Cayman Chemical (Ann Arbor, MI). The Rho kinase inhibitor Y-27632, the phosphodiesterase inhibitor Ro20-1724, protein kinase A, protein kinase A inhibitor and Cellosolve were from Sigma (Saint Louis, MO). Cell culture supplies were from Fisher scientific (Hanover Park, IL). Human platelet concentrates were from Life Source Blood Services (Glenview, IL). [3H]cAMP was from

Binding and signaling of 8-iso-PGF through TPRs

In order to fully characterize the biology of 8-iso-PGF and to address previous inconsistencies concerning its signaling properties [10], [14], we first investigated its binding capacity to TPRs and its functional activation of these receptors using a stable cell line expressing TPRs. Our initial studies demonstrated that the non-TPR transfected HEK cells neither bound [3H]8-iso-PGF nor exhibited detectable calcium (Ca2+) mobilization in response to 8-iso-PGF (5 μM; not shown). Next, we

Discussion

Despite ample evidence supporting a potential role for isoprostanes in disease processes such as apoptosis, brain cell damage, and thrombosis, their biological activity and signaling mechanisms are poorly understood. Nevertheless, certain evidence has suggested that 8-iso-PGF can signal through TPRs. For example, the use of TPR antagonists provided evidence that 8-iso-PGF stimulates platelet shape change and/or weak aggregation through a TPR-mediated process [14], [15], [23]. In contrast,

Acknowledgments

This work was supported in part by a grant from the National Institutes of Health HL23540-24 (to G.C.L.). F.T.K. is a recipient of a predoctoral fellowship from the American Heart Association (Grant 0515533Z). The authors would like to thank Dr. Yee-Kin Ho for his valuable comments and analysis of the data, and Lanlan Dong for technical assistance.

This work has been approved by Ethics Committee at the University of Illinois at Chicago.

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1

Current address: Department of Pharmaceutical Sciences, College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766, United States.

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