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Epoxyeicosatrienoic Acid Relaxing Effects Involve Ca²⁺-Activated K⁺ Channel Activation and CPI-17 Dephosphorylation in Human Bronchi

Le Bilarium, Department of Physiology and Biophysics; Service of Thoracic Surgery; Department of Pathology, Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke, Quebec, Canada

Correspondence and requests for reprints should be addressed to Eric Rousseau, Le Bilarium, Department of Physiology and Biophysics, Faculty of Medicine and Health Sciences, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, PQ, J1H 5N4 Canada. E-mail: eric.rousseau{at}usherbrooke.ca

The aim of the present study was to provide a mechanistic insight into how 14,15-epoxyeicosatrienoic acid (EET) relaxes organ-cultured human bronchi. Tension measurements, performed on either fresh or 3-d–cultured bronchi, revealed that the contractile responses to 1 µM methacholine and 10 µM arachidonic acid were largely relaxed by the eicosanoid regioisomer in a concentration-dependent manner (0.01–10 µM). Pretreatments with 14,15-epoxyeicosa-5(Z)-enoic acid, a specific 14,15-EET antagonist, prevented the relaxing effect, whereas iberitoxin pretreatments (10 nM) partially abolished EET-induced relaxations. In contrast, pretreatments with 1 µM indomethacin amplified relaxations in explants and membrane hyperpolarizations triggered by 14,15-EET on airway smooth muscle cells. The relaxing responses induced by 14,15-EET were likely related to reduced Ca²⁺ sensitivity of the myofilaments, because free Ca²⁺ concentration–response curves performed on -escin–permeabilized cultured explants were shifted toward higher [Ca²⁺] (lower pCa²⁺ values). 14,15-EET also abolished the tonic responses induced by phorbol-ester-dybutyrate (PDBu) (a protein kinase C [PKC]–sensitizing agent), on both fresh (intact) and -escin–permeabilized explants. Western blot analyses, using two specific primary antibodies against CPI-17 and its PKC-dependent phosphorylated isoform (p-CPI-17), confirmed that the eicosanoid interferes with this intracellular process. These data indicate that 14,15-EET hyperpolarizes airway smooth muscle cells and relaxes precontracted human bronchi while reducing Ca²⁺ sensitivity of fresh and cultured explants. The intracellular effects are related to a PKC-dependent process involving a lower phosphorylation level of CPI-17.

Key Words: calcium sensitivity • CPI-17 • epoxyeicosatrienoic acid • membrane potential • organ culture

CLINICAL RELEVANCE The current research and the results obtained may be highly relevant for human diseases such as asthma and chronic obstructive pulmonary disease.