De novo nitric oxide synthesis drives tactile hypersensitivity induced by ATP-sensitive potassium channel opening in mice: relevance to migraine and other headache disorders.

ABSTRACT: Adenosine triphosphate-sensitive potassium (KATP) channel opener levcromakalim is a potent inducer of vasodilation, headache, and migraine attacks in humans and tactile hypersensitivity in mice. Other migraine-inducing agents, such as nitric oxide (NO) donors, calcitonin gene-related peptide, and pituitary adenylate cyclase-activating polypeptide, are thought to activate second messengers leading to KATP opening. However, the mechanism by which KATP channel opening leads to migraine remains unclear. In this study, we investigated the contribution of nitric oxide synthase (NOS) isoforms and downstream signaling cascades in a mouse model of migraine-relevant tactile hypersensitivity induced by repeated administration of levcromakalim. The nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) effectively prevented levcromakalim-induced hypersensitivity. Gene expression analysis in the dura mater suggested contributions from endothelial NOS (eNOS) and inducible NOS (iNOS). Semi-selective neuronal NOS (nNOS) inhibition with S-methyl-L-thiocitrulline or genetic deletion of neuronal NOS had minimal effects on hypersensitivity and no effect on vasodilation. By contrast, eNOS-/- mice were partially protected from levcromakalim-induced hypersensitivity and exhibited impaired vascular response, highlighting eNOS as a key mediator. Inhibition of iNOS with S-methylisothiourea revealed a possible contribution from iNOS as well. Surprisingly, inhibition of soluble guanylate cyclase had no effect, while the peroxynitrite decomposition catalyst FeTPPS partially attenuated hypersensitivity, implicating nitrosative stress - rather than classical NO-soluble guanylate cyclase-cGMP signaling - as the critical downstream pathway. We propose that levcromakalim induces both coupled and uncoupled eNOS activity, enhanced NO production, and generation of reactive nitrogen species, including peroxynitrite. Our findings reveal a pivotal role for eNOS and peroxynitrite in KATP channel-induced migraine-relevant hypersensitivity and support the targeting of nitrosative stress as a potential therapeutic strategy.