Distribution of luciferin-dependent bioluminescence in cardiovascular tissue from <i>Cox2</i>^{<i>fLuc/+</i>} mice.

<p>(a) Quantification of basal expression from the aortic tree, vena cava, chambers of the heart and, for comparison, brain from <i>Cox2</i>^{<i>fLuc/+</i>} mice and (b) and representative images of bioluminescence. Arteries, veins and chambers of the heart were essentially devoid of expression from the <i>Cox2</i> gene, in comparison with the brain as a reference tissue. The only exception to this was weak, but detectable, expression in the region of the aortic arch. n=3.</p

6-keto-PGF_1α production in isolated mouse aorta; measurement by enzyme immunoassay, radio immunoassay, and liquid chromatography tandem mass spectrometry (LC-MS/MS).

<p>Prostacyclin release by isolated rings of mouse aorta stimulated with Ca²⁺ ionophore A23187 (50µM), measured as the stable breakdown product 6-keto-PGF_1α, was not altered by <i>Cox2</i> gene deletion, but was reduced >10-fold by <i>Cox1</i> gene deletion. The pattern and level of 6-keto-PGF_1α accumulation was similar whether measured by (a) enzyme immunoassay, (b) radio immunoassay or (c) LC-MS/MS. Representative LC-MS/MS chromatograms show the presence or absence of 6-keto PGF_1α in all sample types (retention time 2.81 min; transition ion <i>m</i>/<i>z</i> 369>163). n=4-7. *, p<0.05 by 1-way ANOVA with Bonferonni’s post-test.</p

Bradykinin-stimulated prostanoid accumulation in the circulation <i>in</i><i>vivo</i> in wild-type, <i>Cox1</i>^<i>-/-</i>, and <i>Cox2</i>^<i>-/-</i> mice.

<p>Accumulation of the stable prostacyclin breakdown product, 6-keto-PGF_1α in plasma after bradykinin administration (100nmol/kg i.v.) is dependent on COX-1 but not COX-2 when measured by LC-MS/MS (a). Representative LC-MS/MS chromatograms show the presence or absence of 6-keto PGF_1α in all sample types (retention time 2.81 min; transition ion <i>m</i>/<i>z</i> 369>163). Similar data were obtained for plasma levels of PGE₂ (b), 13,14-dihydro-15-keto-PGE₂ (c), PGD₂ (d), TXB₂ (e) and (f) PGF_2α. Plasma 6-keto-PGF_1α levels in all genotypes compare well with those previously published using enzyme immunoassay measurements. n=6. *, p<0.05 by 1-way ANOVA with Bonferonni’s post-hoc test.</p

Distribution of luciferin-dependent bioluminescence in tissues from <i>Cox2</i>^{<i>fLuc/+</i>} mice.

<p>(a) Basal expression from organs of the <i>Cox2</i>^{<i>fLuc/+</i>} mice was visualized by bioluminescent imaging of tissues dissected from <i>Cox2</i>^{<i>fLuc/+</i>} reporter mice after injection of D-luciferin in vivo (125mg/kg i.p.). (b) Imaging data are expressed as maximum luminescent emission from each tissue. Basal <i>Cox2</i> gene driven luciferase expression was present in many tissues including the vas deferens, brain, intestine, and thymus but was notably low to absent in the aorta (highlighted with red circles). Sub-division of the (c) brain, (d) intestine, (e) kidney and (f) stomach revealed regional expression patterns within each tissue. n=5.</p

COX-2-dependent prostanoid production by aorta versus other mouse tissues in <i>Cox1</i>^<i>-/-</i> mice.

<p>(a) PGE₂ formation, normalized to tissue mass, was measured by immunoassay in supernatants of Ca²⁺ ionophore A23187 (50µM)-stimulated tissue segments from <i>Cox1</i>^<i>-/-</i> mice. Cox1^-/- tissues released a variable amount of PGE₂ with low levels in the aorta (highlighted in red), and substantially higher levels in the thymus, intestines, renal medulla, brain and vas deferens. This distribution correlates well with luciferase expression in organs of the <i>Cox2</i>^{<i>fLuc/+</i>} mouse, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069524#pone-0069524-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069524#pone-0069524-g004" target="_blank">4</a>. n=6.</p