117: Nitric oxide drives KATP channel�induced tactile hypersensitivity in mice

Topic: Basic Science
Subtopic: Pain category (ICD-11), Acute headache, , , , , , , , , , Assessment and Measurement of Pain/Nociception, Transgenic models, Behavioral studies

Background and Aims: ATP‑sensitive potassium (KATP) channel opener levcromakalim is a robust inducer of vasodilation (1), headache, and migraine attacks in humans (2), and produces migraine‑relevant tactile hypersensitivity in mice (3). Although other migraine‑inducing agents — such as nitric oxide (NO) donors, CGRP, and PACAP — are thought to activate intracellular second‑messenger signaling that ultimately leads to KATP channel opening (4), the mechanistic link between KATP activation and migraine remains poorly understood. The aim of this study was to investigate the contribution of nitric oxide synthase (NOS) isoforms and downstream signaling pathways in a mouse model of levcromakalim‑induced tactile hypersensitivity.

Methods: We examined the effects of repeated levcromakalim administration on tactile hypersensitivity in mice using von Frey filaments. To evaluate the involvement of specific NOS isoforms and downstream signaling pathways, we applied pharmacological inhibitors (NG-nitro-L-arginine methyl ester (L‑NAME), S-methyl-L-Thiocitrulline (SMTC), S-methylisothiourea (SMT), ODQ, and FeTPPS) and used neuronal NOS (nNOS) knockout and endothelial NOS (eNOS) knockout mice. Gene expression assays were performed on dura mater, trigeminal ganglia, and cerebral blood vessels to assess NOS contributions across tissues. In addition, vascular responses to levcromaklim in WT, nNOS KO and eNOS KO mice were analyzed using wire myography.

Results: Non-selective NOS inhibitor, L‑NAME, effectively prevented levcromakalim‑induced tactile hypersensitivity, while gene expression analysis indicated roles for both eNOS and inducible NOS (iNOS). Neither pharmacological inhibition nor genetic deletion of nNOS substantially affected hypersensitivity or vasodilation. in contrast, eNOS KO mice displayed partial protection from hypersensitivity and impaired vascular responses, identifying eNOS as a key mediator. SMT treatment suggested an additional contribution of iNOS. Notably, sGC inhibition (ODQ) failed to alter hypersensitivity, while FeTPPS partially reduced it, implicating nitrosative stress, including peroxynitrite formation, rather than classical NO‑dependent sGC–cGMP signaling, as the relevant downstream mechanism.

Conclusions: Our findings indicate that levcromakalim activates both coupled and uncoupled eNOS, enhancing NO production and promoting the formation of reactive nitrogen species such as peroxynitrite. These results highlight a central role for eNOS‑derived nitrosative stress in KATP channel‑induced tactile hypersensitivity, providing new insight into the mechanisms linking KATP channel opening to migraine. Targeting nitrosative stress pathways, particularly peroxynitrite, may therefore represent a potential therapeutic strategy for migraine or possibly other types of headache.

References: 1) Ploug KB, Sørensen MA, Strøbech L, Klaerke DA, Hay‑Schmidt A, et al. KATP channels in pig and human intracranial arteries. Molecular and Cellular Pharmacology. 2008

2) Al-Karagholi MAM, Hansen JM, Guo S, Olesen J, Ashina M. Opening of ATP-sensitive potassium channels causes migraine attacks: A new target for the treatment of migraine. Brain. 2019

3) Christensen SL, Rasmussen RH, La Cour S, Ernstsen C, Hansen TF, et al. Smooth muscle ATP-sensitive potassium channels mediate migraine-relevant hypersensitivity in mouse models. Cephalalgia. 2022

4) Al‑Hassany L, Boucherie DM, Creeney H, van Drie RWA, Farham F, et al. Future targets for migraine treatment beyond CGRP. J Headache Pain. 2023

Presenting Author: Rikke Holm Rasmussen
Submitting Author: Rikke Holm Rasmussen

Senior Author: Sarah Louise Christensen

IASP 2026 Global Year theme of Neuropathic Pain: No

• Charlotte Ernstsen: PhD - Danish Headache Center
• Anja Holm: PhD - RNA Therapeutics, Copenhagen University Hospital
• Sabrina Prehn Lauritzen: MSc - Danish Headache Center
• Karina Obelitz-Ryom: PhD - Danish Headache Center
• David Møbjerg Kristensen: PhD - Department of Science and Environment, Roskilde University
• Inger Jansen-Olesen: PhD - Danish Headache Center
• Jes Olesen: Professor - Danish Headache Center
• Sarah Louise Christensen: PhD - Danish Headache Center