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

Adenosine triphosphate-sensitive potassium (K ATP ) 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 K ATP opening. However, the mechanism by which K ATP 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 N G -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 K ATP channel-induced migraine-relevant hypersensitivity and support the targeting of nitrosative stress as a potential therapeutic strategy.