LC-MS/MS Confirms That COX-1 Drives Vascular Prostacyclin Whilst Gene Expression Pattern Reveals Non-Vascular Sites of COX-2 Expression

Abstract

There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.

Age-associated alterations in constitutively expressed cyclooxygenase-2 immunoreactivity and protein levels in the hippocampus

“…COX-2 is an inducible inflammatory mediator, which is constitutively expressed in the brain, kidney, gut and thymus (2). In particular, the constitutive expression of COX-2 is enriched in the hippocampus and cortex (3).…”

“…Among these, COX-2 has attracted the majority of attention, as it is induced by inflammation. COX-2 is constitutively expressed in specific organs, including the brain, thymus, gut and kidneys (2), and constitutive expression of COX-2 in the brain is considered to serve a major role in synaptic plasticity, with prostaglandins generated by COX-2 modulating local cerebral blood flow and learning (3)(4)(5)(6). Previous studies have demonstrated that treadmill exercise significantly increases neurogenesis and COX-2 immunoreactivity in the rat hippocampus (7,8).…”

Fractals and self-organized criticality in anti-inflammatory drugs

“…The molecular mechanism for the cardiovascular toxicity of COX-2 inhibitors is unclear, with some workers ascribing it primarily to COX-2 driving vascular prostacyclin release [18], while others claim that vascular prostacyclin production in healthy blood vessels is generally driven by COX-1 [19,20]. The bicyclic structure of COX-2 identified in Fig.…”

Vasoconstrictor role of cyclooxygenase-1-mediated prostacyclin synthesis in non-insulin-dependent diabetic mice induced by high-fat diet and streptozotocin

“…In fact, the vasomotor reaction to PGI 2 may be modulated by the functionally opposing TP and dilator IP receptors (9,21,47,50), and an EDCF-like action of PGI 2 can result from little or low functional presence of vasodilator IP receptors that leads to a major activation of TP receptors (10,(23)(24)(25)42). Also, COX-2 has been commonly considered a major source of endothelial PGI 2 synthesis (11); however, in some vascular beds, COX-1 functions as the major COX form (16,17), mediating endothelium-dependent contraction (23-25, 39, 41) or relaxation (21,30,38) under normal physiological conditions. On the other hand, the precise role of COX-1 in diseased vessels, such as in diabetes, still remains to be elucidated clearly.…”