HYDROXYL RADICAL DEPENDENT INACTIVATION OF GUANYLATE-CYCLASE IN CEREBRAL ARTERIOLES BY METHYLENE-BLUE AND BY LY83583

Background and Purpose: Methylene blue and 6-anilino,5,8-quinolinedione (LY83583) are used extensively to block activation of guanylate cyclase. Both agents generate oxygen radicals. Therefore, it appeared profitable to investigate whether the generation of oxygen radicals by these agents is responsible for the blockade of responses to nitrodilators that act via activation of guanylate cyclase to relax vascular smooth muscle and cause vasodilation. Methods: We tested in anesthetized cats equipped with cranial windows responses to topical application of nitroglycerin, nitroprusside, and adenosine before and during topical application of methylene blue (5 muM). Responses to the vasoactive agents were tested during application of methylene blue after permeabilization of the cell membrane with a detergent to allow methylene blue to enter vascular smooth muscle. Responses were also tested in the presence of superoxide dismutase, catalase, deferoxamine, or dimethyl sulfoxide to scavenge reactive products of oxygen metabolism or to eliminate catalytic iron. In additional experiments we tested the effects of topical application of nitroprusside or adenosine before and after application of LY83583. The responses to the vasoactive agents were also tested in the presence of superoxide dismutase, catalase, or dimethyl sulfoxide in addition to LY83583. We also tested responses to calcitonin gene-related peptide before and in the presence of LY83583 with or without superoxide dismutase. Results: Methylene blue eliminated the arteriolar dilation in response to nitroprusside and nitroglycerin after permeabilization of the cell membrane with a detergent but not before. The responses to adenosine were unaffected. The blockade induced by methylene blue was reversed by superoxide dismutase, catalase, or dimethyl sulfoxide but not by deferoxamine. LY83583 blocked responses to nitroprusside but not to adenosine. The blockade was eliminated by superoxide dismutase, catalase, or dimethyl sulfoxide. LY83583 blocked the vasodilation induced by calcitonin gene-related peptide. This blockade was reversed by superoxide dismutase. Conclusions: Methylene blue and LY83583 prevent the activation of soluble guanylate cyclase by nitrodilators or by calcitonin gene-related peptide by generating oxygen radicals. The mediator of this response is the hydroxyl radical. Methylene blue does not enter the vascular smooth muscle of cerebral arterioles unless the cell membrane is permeabilized.