Methylene blue but not changes in cyclic GMP inhibits resting and bradykinin‐stimulated production of prostacyclin by pig aortic endothelial cells

Abstract

1. Primary cultures of pig aortic endothelial cells produced 6-keto-prostaglandin F1 alpha (6-keto PGF1 alpha), the stable breakdown product of prostacyclin, both in the resting state and in response to bradykinin. The rise in 6-keto-PGF1 alpha production induced by bradykinin (1-100 nM) was concentration-dependent. 2. Treating endothelial cells with the inhibitor of soluble guanylate cyclase, methylene blue (0.1-20 microM) produced an irreversible reduction in resting and bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha with an IC50 of 0.5 +/- 0.1 microM. Treating endothelial cells with haemoglobin (10 microM) had no effect on resting or bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha. 3. Two stimuli that elevate the level of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in endothelial cells, 8-bromo cyclic GMP (30 microM) and atriopeptin II (0.1 microM), each had no effect on resting or bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha. Furthermore, treating endothelial cells with either 8-bromo cyclic GMP (30 microM) or atriopeptin II (0.1 microM) had no effect on the ability of methylene blue (20 microM) to inhibit resting or bradykinin (0.1 microM)-stimulated production of 6-keto-PGF1 alpha. 4. Adding arachidonic acid (1 microM) to endothelial cells led to a marked stimulation of 6-keto-PGF1 alpha production. Treating cells with either methylene blue (20 microM) or the cyclo-oxygenase inhibitor, flurbiprofen (10 microM), inhibited both resting and arachidonic acid (1 microM)-induced production of 6-keto-PGF1 alpha. 5. In pig aortic endothelial cells methylene blue appears to block prostacyclin production by a mechanism independent of inhibition of soluble guanylate cyclase. Care should be exercised when using methylene blue as a selective inhibitor of endothelium-derived relaxing factor due to its additional ability to block production of the other endothelium-derived vasodilator, prostacyclin.