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

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Abstract
1 Primary cultures of pig aortic endothelial cells produced 6-keto-prostaglandin F1α (6-keto PGF1α), the stable breakdown product of prostacyclin, both in the resting state and in response to bradykinin. The rise in 6-keto-PGF1α 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 μm) produced an irreversible reduction in resting and bradykinin (0.1 μm)-stimulated production of 6-keto-PGF1α with an IC50 of 0.5 ± 0.1 μm. Treating endothelial cells with haemoglobin (10 μm) had no effect on resting or bradykinin (0.1 μm)-stimulated production of 6-keto-PGF1α. 3 Two stimuli that elevate the level of guanosine 3′:5′-cyclic monophosphate (cyclic GMP) in endothelial cells, 8-bromo cyclic GMP (30 μm) and atriopeptin II (0.1 μm), each had no effect on resting or bradykinin (0.1 μm)-stimulated production of 6-keto-PGF1α. Furthermore, treating endothelial cells with either 8-bromo cyclic GMP (30 μm) or atriopeptin II (0.1 μm) had no effect on the ability of methylene blue (20 μm) to inhibit resting or bradykinin (0.1 μm)-stimulated production of 6-keto-PGF1α. 4 Adding arachidonic acid (1 μm) to endothelial cells led to a marked stimulation of 6-keto-PGF1α production. Treating cells with either methylene blue (20 μm) or the cyclo-oxygenase inhibitor, flurbiprofen (10 μm), inhibited both resting and arachidonic acid (1 μm)-induced production of 6-keto-PGF1α. 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.