Indomethacin potentiates acetylcholine-induced vasodilation by increasing free radical production.

1 We studied the effects of indomethacin on endothelium-dependent and -independent vascular relaxation in rat thoracic aortic rings and its role in superoxide anion (O2 -) production. 2 We measured isometric force changes in response to acetylcholine (Ach, 1 nM–0.1mM), sodium nitroprusside (SNP, 0.1 nM–0.1 microM; a nitric oxide (NO) donor) and cromakalim (1 nM–0.1mM; a KATP-channel opener) in aorta rings contracted with norepinephrine (NE, 0.1 microM). Indomethacin (10 microM; 20 min) significantly increased Ach-induced vasodilation (EC50 decreased from 8.99 microM to 16 nM). The free radical scavengers superoxide dismutase and 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl completely reverted these effects. Indomethacin did not affect SNP- or cromakalim induced vasodilation. 3 Neither acetylsalicylic acid (ASA, 5–100 microM; 15 min) nor ketoprofen (1-100 microM; 15 min) affected Ach, SNP and cromakalim concentration–response curves. 4 Incubation of the aorta with Ach (1 microM) rapidly and markedly increased intracellular NO fluorescence in the aorta endothelium. Indomethacin did not affect Ach-induced NO production. 5 We measured intracellular O2- in the aorta endothelium with dihydroethidium (DHE) dye. Indomethacin significantly increased O2- fluorescence versus controls. Neither ASA nor ketoprofen affected O2- fluorescence. 6 Nitrotyrosine staining was increased in indomethacin-treated aorta sections exposed to Ach, which indicates endogenous formation of peroxynitrite. It was low in aorta sections exposed to Ach alone or with ASA or ketoprofen. 7 We cannot judge if indomethacin-induced endothelium-dependent vasodilation damages or protects the cardiovascular system. Here, we show that indomethacin acts on the cardiovascular system regardless of cyclooxygenase inhibition.