LC-MS/MS Confirms That COX-1 Drives Vascular Prostacyclin whilst Gene Expression Pattern Reveals Non-Vascular Sites of COX-2 Expression.
Kirkby, N.S. Zaiss, A.K. Urquhart, Paula Jiao, J. Austin, P.J. Al-Yamani, M. Lundberg, M.H. MacKenzie, L.S. Warner, T.D. Nicolaou, Anna
Kirkby, N.S.
Zaiss, A.K.
Urquhart, Paula
Jiao, J.
Austin, P.J.
Al-Yamani, M.
Lundberg, M.H.
MacKenzie, L.S.
Warner, T.D.
Nicolaou, Anna
Publication Date
2013-07-09
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2013-06-07
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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.
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Kirkby NS, Zaiss AK, Urquhart P, Jiao J, Austin PJ, Al-Yamani M, Lundberg MH, Mackenzie LS, Warner TD, Nicolaou A, Herschman HR and Mitchell JA (2013) LC-MS/MS Confirms That COX-1 Drives Vascular Prostacyclin Whilst Gene Expression Pattern Reveals Non-Vascular Sites of COX-2 Expression. PLoS One, 8 (7): e69524.
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